AbstractΔ5 and Δ6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentanoic and docosahexanoic acids. They are encoded by distinct genes in mammals and in Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Δ6 desaturase genes. The 1590 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b5 domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisae it conferred the ability to convert linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) to their corresponding Δ6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition, it conferred on the yeast the ability to convert di-homo-γ-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentanoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Δ5 and Δ6 desaturase activity. This is the first report of a functionally characterized desaturase enzyme of fish, and the first report of a fatty acid desaturase in any species with both Δ5 and Δ6 activity. The zebrafish Δ5/Δ6 desaturase may represent a component of a prototypic vertebrate PUFA biosynthesis pathway.